text:
Castle (disambiguation)
Castle (District Electoral Area)
Castle (novel)
& (disambiguation)
* (disambiguation)
承德 (disambiguation)
Castles are fortified residences built in Europe and the Middle East in the Middle Ages.
Castle or Castles may refer to:
Castle, Bedford, an electoral ward in Bedford, England
Castle, Cardiff, an administrative community in the Welsh capital
Castle, Leicester, an electoral ward in Leicester, England
Castle (Colchester electoral ward), an electoral ward in Colchester, England
Castle (District Electoral Area), Belfast, Northern Ireland
Castle, Cambridge, a district and electoral ward of the city of Cambridge, England
Castle, Newcastle upon Tyne, a ward of the city of Newcastle upon Tyne, England
Castle, Swansea, an electoral ward in Swansea, Wales
Castle, Oklahoma, a town in the United States
Castle Geyser in the Upper Geyser Basin of Yellowstone
Newcastle-under-Lyme or Castle
The Smithsonian Castle, Washington, DC, USA
Castle (novel), a 2000 novel by Garth Nix
Castle (book), a 1978 book by David Macaulay
Castle (card game), designed by Bruno Faidutti and Serge Laget
This page contains text from Wikipedia, the Free Encyclopedia -	https://wn.com/Castle_(disambiguation)
Castle is one of the ten district electoral areas in Belfast, Northern Ireland. Located in the north of the city, the district elects six members to Belfast City Council and contains the wards of Bellevue, Cavehill, Chichester Park, Duncairn, Fortwilliam and Innisfayle. Castle, along with the neighbouring Court and Oldpark districts and parts of Newtownabbey Borough Council, forms the Belfast North constituencies for the Northern Ireland Assembly and UK Parliament. The district is bound to the east by the Victoria Channel, to the north by Newtownabbey Borough Council and Belfast Lough, to the south by North Street and to the west by the Cavehill Road and Oldpark Road.
The district takes its name from Belfast Castle which is located on Cavehill in the north of the district, while the southern section of the district is part of the city centre and forms one of the Belfast's main cultural areas, known as the Cathedral Quarter. The east of the area also contains the Port of Belfast. Castle is served by the M2 and M5 motorways and the Yorkgate railway station.
This page contains text from Wikipedia, the Free Encyclopedia -	https://wn.com/Castle_(District_Electoral_Area)
Castle is the second book in Garth Nix's The Seventh Tower series, published in October 1, 2000 by Scholastic. The cover design and art are by Madalina Stefan and Steve Rawlings respectively.
Tal and Milla make it from the shadowy 'Dark World' to the titular castle, a seeming place of peace. Both are unwanted by the castle's inhabitants, Milla the most. The two must avoid conspiracies and other dangers inside the castle, just to survive.
This page contains text from Wikipedia, the Free Encyclopedia -	https://wn.com/Castle_(novel)
&, or ampersand, is a typographic symbol.
& may also refer to:
& (Ayumi Hamasaki EP)
& (The Moth & the Flame EP)
Iain Baxter&
This page contains text from Wikipedia, the Free Encyclopedia -	https://wn.com/&_(disambiguation)
The symbol * is called asterisk (42 in ASCII). The symbol may also refer to:
"*", a song by M83
"*: Asterisk", a.k.a. "Asterisk (song)", by Orange Range
"*", a song by Sadist from Lego
*, the logo for the alternative rock band the Red Hot Chili Peppers
A symbol for not out in cricket
A symbol identifying a genetic lineage as a paragroup of a specified haplogroup
Star (game theory), the value given to the game where both players have only the option of moving to the zero game
In linguistics, a symbol that prefixes a word or phrase that, in historical linguistics, is a reconstructed form for which no actual examples have been found; and in linguistics of a modern language (see: synchronic linguistics), is judged ungrammatical
The symbol is used to refer a reader to a footnote
The symbol is used to refer a reader to an endnote
In telephony, the beginning of a Vertical service code
Asterisk (disambiguation)
This page contains text from Wikipedia, the Free Encyclopedia -	https://wn.com/*_(disambiguation)
承德 could refer to:
Chengde (承德市), prefecture-level city in Hebei Province, PR China
Chengde County (承德县/承德縣), a county of Chengde City, Hebei Province, PR China
Jōtoku (承德), a Japanese era name
This page contains text from Wikipedia, the Free Encyclopedia -	https://wn.com/承德_(disambiguation)
Related Sites Copy
stcastleguildo.com
balmoralcastles.com
castleking.com
balmoralcastle.org
castleresourcess.com
balmoralcastle.net
festivalcastle.com
newcastleradio.com
acastle.org


Skillshare is a Transition initiative encouraging the community to share simple skills. Community is at the heart of Transition, with the belief that creating happier, fairer and stronger communities will in turn support us to begin to build a more sustainable society.
Join our online community to receive regular updates on when the next skillshare event will take place.
Skillshares are mostly free and take place all over St Andrews. If you would like to offer a skill to share then please feel free to post in the group or email Tansy on tt39@st-andrews.ac.uk. If you have an idea of something you might like to learn, then please post it and we will see who we can find to help.


anyone on here own one?
easy (and FUN!!) to patch up with two Borg filters and one Joystick module.
that was my thinking too. still cool nonetheless.
i've known about his DIY page for awhile but barely skimmed it. what was i thinking.
yup, I think that was the first 1200 series module. I think only 9 were made, and Grant said he had a hard time selling those! Was $209-- if I recall.
It has the same PCB as Wiard Borg-- just not totally populated-- the two VCO/LFOs on the board are empty.
Still have mine it sounds great, of course, but can get out of control easily.
do you have any outta control soundclips?
hmmmm.... i'll try and find some clips, or make. As far as out of control--well that part isn't too exciting... nor accurate I guess. It's just tough to get it from jumping to seriously loud and piercing self oscillation--esp. since you're controlling 2 filters at once, and it lacks any adjustments of the joystick XY levels.
The Doepfer A-101-2 is similar and a bit rougher/less woody sounding and a bit more controllable.
cool. i was just joking about the outta control. just really curious to here any wiard stuff right now. especially discontinued things.
Woah, that sounds pretty cool - plus you gotta love a Dune reference (even if it's just a movie reference).
it doesn't wanna work for me. i'll try back later.
ooh that joyrider looks cool with davies in blue.
"violating Nyquist for yr listening/dancing pleasure."
are the 2 filters in series? or parallel?


In verses such as Noach 8:1 ("And God remembered Noah and all the beasts and all the cattle that were with him in the ark, and God caused a spirit to pass over the earth, and the waters subsided"), what does it mean for God to "remember" something? Does this not imply that He "forgot" something? What does it mean for God to be "reminded" of something?
I am looking for sources that explicitly discuss this topic.
And saving creatures from something that afflicts them, this is called "remembrance". As in his statement: "And God remembered Noah" (Gen. 8:1), "and God remembered Rachel" (Gen. 30:22).
R. Elijah Mizrahi similarly writes (Gen 19:29 s.v. k'shehaya) that remembering refers to saving. See also Hovot HaLevavot (Sha'ar HaYihud: 10).
ויזכור אלהים - חלילה להיות שכחה לקטון שבמלאכי השרת, אף כי ליוצר הכל, שאין שכחה נגדו... רק כאשר תראה טובת השם בארץ ידבר הכתוב, כי זכר אשר הטיב לו בא לפני השם.
And God remembered: heaven fore-fend that forgetfulness apply to the least of [God's] ministering angels, let alone to the Creator of everything, for there is no forgetfulness that can apply to him...Just that when God's goodness is manifested in the world, the verse will describe it as a remembrance of goodness done to God, coming to him.
Radak writes very similarly (ibid). He applies the principle that "the Torah speaks in the language of Man", (cf. Berakhot 31b, Moreh Nevukhim 1:29) to explain that the Torah uses terminology familiar to humans in reference to God, even though their most literal meaning doesn't apply to him. He uses this to similarly explain "But I will for their sakes remember the covenant of their ancestors" (Leviticus 26:45), and related verses.
This 18th century commentary of R. Naftali Hertz Wessley cites support for such explanations from Genesis (40:29) "Yet the chief butler did not remember Joseph, but forgot him". Certainly, he asserts, the butler often remembered his time in prison and his experience with Joseph there. The term "remembering" in that context, therefore refers to focusing on something's characteristics, in order to respond positively or negatively to something. The butler ignored Joseph, so he is said to have "forgotten". Similarly, when God is said to forget or remember, it refers to his behaviour.
Why do you forget us forever; forsake us for the length of days.
The structure of the verse parallels לָנֶצַח with לְאֹרֶךְ יָמִים (terms for eternity), and תִּשְׁכָּחֵנוּ (forgetting us) with תַּעַזְבֵנוּ (abandoning us).
The bible is using a language we humans can understand. G- d does not forget anything. All our lives are recorded, even every word we have spoken. But He will not remember our sins,if they are forgiven (Isa 43:25). "I will remember their sin no more" (Jer 31:34). When G-d "remembers" it is often related to His covenants He made. With Noah (Gen 9:15+16) an everlasting covenant: with Abraham, Isaac and Jakob (Exo 32:13) BTW this covenant includes the land,"this land will I give to your seed and they shall inherit it for ever" "and I will remember the land" (Lev 26:42). When G-d remembers according to His word, it can be for salvation, but also for judgment. "And they consider not, that I remember all their wickedness" (Hos 7:2) But in spite of this G-d will remember His covenant with Abraham and establish an everlasting covenant (Eze 16:60), where the people will not be taught, but will know G-d personally (Jer 31:31-34).
Not the answer you're looking for? Browse other questions tagged hashkafah-philosophy tanach theology parashat-noach memory .


At the luxury KC Beach Club, Koh Samui, we pride ourselves not only on the quality of our food and impeccable service but also on the unique environment of the dining experience itself.
Due to the many and varied dining options available at the KC Beach Club, dining can be a romantic, private or personal occasion depending on your individual tastes and requirements.
The KC Beach Club intends to continue raising the standards for fine dining on the island of Koh Samui. By boasting a wine list with a selection of over three hundred unique labels from the most famous wine growing regions of the world, this luxury hotel will continue to build on its reputation for providing guests with both unforgettable dining and wining experiences.
Overlooking the white powdery sands of Chaweng beach and its azure waters, rooftop of NEO Restaurant is the picturesque spot you have always dreamed of in the post cards.


Previously in this blog we've covered all of the work that NEC Foundation of America does to support Science, Technology, Engineering and Mathematics (STEM) learning in the communities where we live and work.
Last week, NEC witnessed first-hand how the foundation's hard work in the STEM arena is paying off when a delegation attended the ribbon-cutting ceremony and grand opening of the computer lab at Irma Rangel Young Women's Leadership School in Dallas.
Among the delegates and speakers at the event were Dallas ISD Superintendent, Michael Hinojosa, Young Women's Preparatory Network CEO, Lynn McBee, NEC Corporation of America CIO, Juan Fontanes and Lesly Zamora, an Irma Rangel student who interned in the IT department at NEC Corporation of America this past summer.
Dr. Hinojosa stressed the importance of Irma Rangel and applauded Principal Lisa Curry for her role in helping fill the jobs pipeline for young women in STEM careers. He also noted the fact that the school's success is made possible by the Young Women's Preparatory Network, and community support from donor organizations like NEC Foundation of America.
Meanwhile, Mr. Fontanes outlined a brief history of NEC's relationship with Irma Rangel and the Young Women's Preparatory Network. After a tour of the campus earlier this year, NEC donated dollars, employee time and IT expertise to redesign and procure new equipment for the Irma Rangel computer lab. The new lab sports 26 new Dell computers, updated furniture and an interactive projector at the front of the lab.
Event attendees, including local dignitaries and media, were also treated to a demonstration by Ms. Zamora, who showcased her ability to tear down and rebuild a computer in just six minutes. The Dallas Morning News has posted a time-lapse video of the impressive demo on their blog.
Check out more photos, videos and media coverage from the event on our Facebook page.
Posted by Troy Marsh on February 25, 2016 February 26, 2016 Posted in Education	Tagged Clay Jenkins, DISD, education, irma rangel, NEC Foundation, S.T.E.M.
DALLAS – NEC Corporation of America President and CEO Shin Takahashi accompanied DISD Superintendent Michael Hinojosa and Dallas County Judge Clay Jenkins during a tour of several S.T.E.M. programs at middle and high school campuses on Wednesday, February 24 2016. The tour provided an opportunity to see students in action and learn the positive impact S.T.E.M. education has on student achievement.
The NEC Foundation of America is investing $250,000 in the S.T.E.M. program of the Irma Rangel Young Women's Leadership School and will be recruiting two interns from the senior class. S.T.E.M. education is one of the main focus areas of the Foundation's mission to contribute to the creation of social value and advance society through innovative giving programs. District leaders say they are working to expand S.T.E.M. initiatives in more middle schools over the next three years.
Hey Buddy!, I found this information for you: "American Ingenuity and NEC Technology Combine to Provide the Cape Cod area with Improved Service". Here is the website link: http://nectoday.com/american-ingenuity-and-nec-technology-combine-to-provide-the-cape-cod-area-with-improved-service/. Thank you.


New York City's recent fickle weather has been tough for everyone, but it's even worse on those of us who grew up with the warm, consistent weather of Los Angeles. I usually get homesick every winter — it's especially bad after I talk to my parents and find out the temperature was 80 degrees there in the middle of the winter, while I'm here, freaking out about wind-chill advisories — but there are times all year round when being a Californian in New York is a challenge. When people ask, "Why would you ever leave southern California?" I know that it's a valid question.
Don't get me wrong, I love New York City. There are plenty of reasons why it's worth living here — from the limitless choices for theater and other cultural activities, to the accessible public transportation, to the range of food options available any time of day or night. I've lived here for seven years now, and in that time, I've adjusted to life in this big, bad city and learned a lot about what it takes to survive here — for example, how to avoid eye contact with anybody who wants to know if you like comedy, says they have a question about your hair, or asks where all the best public bathrooms are. It's a skill.
But I'll never forget my roots — I still consider myself a Californian. So whether you just moved here or have lived here for years, if you'll always consider yourself an Angeleno, even while you're running for a rush hour 6 train, here are some things that you'll understand.
You can get anywhere on the subway and you don't have to worry about parking! Look at all the pretty lights in Times Square!
Central Park is so beautiful when it's covered in clean, white snow.
Now it's not magical; it just means that you can't wear the shoes you want to wear. And it's cold!
And then you overcompensate by wearing too many layers because you don't know how to dress for any weather that is below 60 degrees.
But at least, now that you're perpetually annoyed, you fit in with the New Yorkers.
If you're touching me, you're too close. How hard is that to understand?
8. You Don't Understand Why Everyone Here Says "On Line" Instead Of "In Line"
When people ask if you are waiting on line, you want to tell them, "No. I'm waiting in line." Online is where you go to send e-mails and look things up. In line is what you do at a grocery store.
When you're walking with your friends and there's a red light, they are already at the other side of the street before they realize that you're still waiting for the green man to appear so you can cross.
But you're still going to eat at Chipotle. Because while it may not be authentic, it tastes delicious and it's cheaper than most places in the city.
But you complain that it is way more expensive than In-N-Out the whole time. And they don't even have animal style fries!
And none of your New Yorker friends understood why. Two words: ice blended.
And you start to give them recommendations for things to do in California, until you realize they mean Disney World.
Now when saying how long it will take you to get somewhere, you add "depending on the trains" instead of "depending on traffic."
Now you complain about having to go from Brooklyn to Queens. The concept of what's far is really different on the subway than in a car.
Finally, someone who understands you taco snobbery!
18. There Are Things You Really Don't Miss About L.A.
Mostly the traffic. And searching for a parking spot.
Only people from L.A. can insult it! And believe me, we've heard it all: everyone in L.A. is superficial or lazy or too health conscious. But we know the truth: people from L.A. are awesome.


text:
Popdust Presents
Astros' Punishment for Cheating Scandal Highlights Pete Rose's Unreasonable Ban
13 Musicians Influenced By Psychedelics
8 Civil Rights Movies to Watch on Martin Luther King, Jr. Day
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Arts 09/07/18
'Beetlejuice' is Coming to Broadway
Weirdness and Wonder are Headed for the Great White Way
Melissa Kay
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Fans of film, especially the freaky kind, better book a trip to the Big Apple.
Early spring 2019 will bring Beetlejuice to Broadway – a musical version, no less – jam-packed with all the fun the movie version filled us with in the late 1980s.
While the cast will be new and the plot made more stage-friendly, the adaptation of Tim Burton's comedy-fantasy flick is sure to be just as crazy-cool as it was on the big screen. We'll wait and see who replaces Winona Rider, Catherine O'Hara, Michael Keaton, Geena Davis, Alec Baldwin and the rest of the kooky crew, but seeing the story play out on stage is sure to surprise and delight.
This sort of story is perfect for live audiences – filled with ghosts with the most and plenty of thrills and chills. With lots of laughs peppered in and considerable costume potential, Beetlejuice on Broadway has all the makings of a hit. Hamilton status? Not likely, but a fun-for-the-family theater experience sure to land rave reviews and a resurgence of Beetlejuice-themed Halloween getups come October.
While fans of the movie are far and wide, if this sort of play isn't your idea of the perfect date night, The New York Times reports, "The announcement comes as the Broadway season is rapidly taking shape — 'Beetlejuice' is the 10th new musical to announce plans for a production, and a few more are expected." Looks like the year to come and beyond will be filled with plenty of Broadway tunes to tap your toes to.
http://www.joblo.com
But for Beetlejuice fans, there is no need to wait 'till the production hits Manhattan. As per The New York Times, "The Washington production of "Beetlejuice" begins performances Oct. 14 and opens Nov. 4 at the National Theater. The cast there is led by Alex Brightman ('School of Rock'), playing the ghost Betelgeuse, and Sophia Anne Caruso ('Lazarus'), playing the living couple's daughter, Lydia." Beetlejuice will begin previews starting March in Manhattan at the famed Winter Garden Theater.
s.yimg.com
In case you are not familiar with Beetlejuice, Entertainment Weekly sums it up best, "As with Tim Burton's beloved movie, the musical follows unusual teen Lydia Deetz, who moves into a house haunted by a recently deceased couple and the titular degenerate demon. Beetlejuice and Lydia then hatch a devilish plot (including exorcism and arranged marriages) to scare away her parents." Sounds weird, but it's weirdly wonderful!
Beetlejuice! Beetlejuice! Beetlejuice!
images.amcnetworks.com
Melissa A. Kay is a New York-based writer, editor, and content strategist. Follow her work on Popdust as well as sites including TopDust, Chase Bank, P&G, Understood.org, The Richest, GearBrain, The Journiest, Bella, TrueSelf, Better Homes & Gardens, AMC Daycare, and more.
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redpajama_set_name: RedPajamaCommonCrawl


TickCheck.com is a university-affiliated lab based in East Stroudsburg, Pennsylvania that is dedicated to providing fast, highly rated, laboratory-quality tick testing to people in high-risk areas for Lyme and other tick-borne diseases nationwide.
We offer PCR-based tick testing that is 99.9% accurate and can detect the presence of 18 pathogens that cause diseases such as Lyme disease, anaplasmosis, babesia, bartonella, ehrlichiosis, tularemia, B. miyamotoi, mycoplasmosis, southern tick-associated rash illness, and Rocky Mountain spotted fever.
Unlike traditional laboratories' complicated test requisition forms, TickCheck.com has a simple, easy to use interface for ordering tick tests. Just order your tests, mail your tick to our East Stroudsburg laboratory, and your results will be ready within 24-48 hours of lab receipt - guaranteed. Got a tick? Get started now!
Jonathan Weber is the founder and CEO of TickCheck. A serial entrepreneur, Jonathan launched TickCheck as a collaboration with his university's Wildlife DNA Laboratory.
Jonathan became acutely aware of the dangers of tick-borne diseases after his father caught Lyme during a family trip on the Appalachian Trail. An avid hiker, he is excited to help bring tick testing services to millions of people for the first time via the Internet.
Nicole Chinnici is the laboratory director of TickCheck. Nicole has over five years of laboratory experience and will be overseeing the tick testing process, from tick identification through PCR testing and quality control.
Nicole first became involved in tick-borne disease research while working as a graduate student. Since then Nicole has used PCR to explore many topics related to both disease and wildlife.
Mendim Bardhi is the lead developer of TickCheck. With over five years of web and network experience, he aids Jonathan Weber in leading the team that manages the TickCheck website and lab interfaces.
Mendim has a passion for modern, intuitive web infrastructures. His motivation for building TickCheck.com was to provide a high-performing interface to a spectactular service. He ensures TickCheck.com runs smoothly every day.


text:
How to Play Family Feud Without Downloading
by Mark Keller
You don't have to download the Family Feud video game to have a great time playing it. With the use of Flash technology, you can play the popular TV quiz game right in your web browser.
Go to a website that has Family Feud available in Flash, such as GameFools.com and Games.com. Some sites may ask you to log in so you can get your name in the scoreboard if you win, but others allow you to play immediately. Click on the page with the Family Feud game, and wait for Flash to load the game data.
Once the game has loaded, press the "Start" button. You'll be taken to the first round of the game, in which you have to guess as many answers to a single question as possible in 20 seconds. The questions are generally trivia or opinion-related, and the answers are those most often given when asked of 100 people. Type in your answer and either press "Submit" or the Enter key on your keyboard. Once you guess wrong three times, or get all the right answers, your score will be tallied and you'll be taken to the next round.
Round two is the same format but with a different, possibly more difficult question. Questions range from things like, "What do people do when given a shot?" to "What is a good gift for a pirate?" When the round is finished, your two scores will be added together.
The third and fourth rounds are played the same, but scores in the third round are doubled, and those in the fourth round are tripled.
The last round of the game is called "Big Money." Instead of multiple answers to one question, you'll be asked five questions over the course of a minute. You'll then be asked the same questions a second time, but you have to give different answers. Your score is based on how many people replied the same way to each question as you. For instance, if your answer to a question is "Baseball," and 60 people answered "Baseball" as well, you gain 60 points. If you manage to get 200 points or more, you'll get 20,000 bonus points and win.
Speakers (optional: for background music and voiceover)
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Gram-negative bacteria provide a particular challenge to antibacterial drug discovery due to their cell envelope structure. Compound entry is impeded by the lipopolysaccharide (LPS) of the outer membrane (OM), and those molecules that overcome this barrier are often expelled by multidrug efflux pumps. Understanding how efflux and permeability affect the ability of a compound to reach its target is paramount to translating in vitro biochemical potency to cellular bioactivity. Herein, a suite of Pseudomonas aeruginosa strains were constructed in either a wild-type or efflux-null background in which mutations were engineered in LptD, the final protein involved in LPS transport to the OM. These mutants were demonstrated to be defective in LPS transport, resulting in compromised barrier function. Using isogenic strain sets harboring these newly created alleles, we were able to define the contributions of permeability and efflux to the intrinsic resistance of P. aeruginosa to a variety of antibiotics. These strains will be useful in the design and optimization of future antibiotics against Gram-negative pathogens.
With emerging multidrug resistance and a limited number of treatment options, bacterial infections pose an ever growing threat to human health. Gram-negative bacteria are particularly recalcitrant to antimicrobial intervention due to their cell envelope structure. Possessing two membranes with different chemical properties (1) and containing an arsenal of efflux pumps (2), Gram-negative bacteria are capable of both excluding and expelling molecules, rendering them resistant to many drugs. Despite major Gram-negative pathogens being classified as urgent or serious threats by the CDC (3), no Gram-negative-active antibiotic with a new mechanism of action has been introduced in over 40 years. Of the 28 new antibiotics approved since 2000, only 18 are indicated for treatment of Gram-negative bacteria (4), and all of those are derived from the four legacy classes β-lactams, tetracyclines, macrolides, and fluoroquinolones, which were first introduced in 1942 (5), 1948 (6), 1952 (7), and 1967 (8), respectively. Novel derivatives of old antibiotics often have limited spectra of coverage and can only do so much to overcome existing mechanisms of resistance; therefore, introduction of novel classes of antibiotics is critical.
There is no shortage of potential targets in the antibacterial space given that the essential gene set for several Gram-negative pathogens has been defined (9–12). However, despite genetically demonstrating target essentiality, cognate inhibitors with exquisite in vitro activity often have no measurable cellular activity (13, 14). The reasons for this can be attributed to a lack of penetration of compounds into bacteria, efflux of molecules out of bacteria, insufficient inhibition of the target, metabolism within the cells, or a combination of the aforementioned factors. Understanding the reason for failure to translate enzymatic 50% inhibitory concentrations (IC50s) into cellular MICs is paramount to an antibacterial research and development program and guides medicinal chemistry efforts. As a model organism for Gram-negative pathogens, many genetic tools have been developed for Escherichia coli in order to assess the contributions of efflux and permeability to intrinsic resistance. Strains exist with efflux pumps knocked out either individually or in combination, and the relative contribution of each system to the susceptibility to major classes of antimicrobials has been defined (15). For the most part, tolC is the major contributor to efflux in E. coli, and knockout of this gene is often used to assess whether novel compounds lack cellular activity due to efflux (16–24). Similarly, mutations in several genes—including lpxC (25–27), lptD (28, 29), and lptE (30, 31)—leading to increased permeability of E. coli have been described, and strains harboring such lesions are often used to assess the effect of increased cellular penetration on the bioactivity of molecules (32–37). These three genes in particular are often targeted because of their essential role in biosynthesis and biogenesis of lipopolysaccharide (LPS), which comprises the outer leaflet of the outer membrane (OM) in Gram-negative organisms and greatly contributes to reduced permeability. LpxC is a zinc-dependent deacetylase that forms UDP-3-O-(hydroxytetradecanoyl)glucosamine in the committed step of lipid A biosynthesis (38), and LptD and LptE constitute a β-barrel and plug, respectively, responsible for the final step of LPS transport to the OM (39–41).
Although E. coli serves as a powerful model for studying Gram-negative bacteria, findings are not always recapitulated in other more serious pathogens, in particular Pseudomonas aeruginosa. P. aeruginosa is a common nosocomial pathogen that often takes advantage of immunocompromised hosts to cause infections associated with high rates of mortality (42, 43). It is perhaps best known as the major cause of declining lung function in cystic fibrosis (CF) patients (44). Among Gram-negative bacteria, P. aeruginosa is particularly resistant to antimicrobial intervention because it has a large and diverse set of multidrug efflux pumps (45, 46) and an especially impermeable OM (47, 48), and it can metabolize antimicrobial compounds (43). Although the permeability of the lipid bilayer domain of P. aeruginosa, as assessed by measuring the influx of hydrophobic steroid probes, is comparable to those of other Gram-negative bacteria such as Salmonella enterica serovar Typhimurium and E. coli (48, 49), a limitation to the influx of molecules comes from OprF, the major porin of P. aeruginosa. Responsible for slow nonspecific diffusion of solutes into the bacterium, it has been shown that OprF folds into two conformations, with less than 5% of the porin present in the open-channel conformer, severely limiting penetration of molecules into the bacterium (50). It is for this multitude of reasons that often agents which are active on most other Gram-negative bacteria are less effective on Pseudomonas aeruginosa (13, 51–53), and finding compounds that inhibit this organism can be difficult. Again, it is imperative to understand the roles efflux and permeability play in the intrinsic resistance of this organism when developing the next generation of antibiotics. As with E. coli, collections of efflux pump-null strains of P. aeruginosa have been created which help to define the substrate specificities of the various pumps (43, 54, 55). However, P. aeruginosa efflux pumps are more numerous and redundant than those of E. coli, and often strains with multiple pump knockouts are necessary to assess the full effect of efflux on compound activity (13, 56–58). In contrast, techniques for assessing the effects of permeability in P. aeruginosa are less refined. Rather than single defined mutations, as utilized in E. coli, membrane permeabilization is often induced with chemical treatment using reagents such as polymyxin B nonapeptide (PMBN), compound 48/80, and EDTA (59–66). The latter two reagents can be harsh, inconsistent, and have incomplete effects, have intrinsic bioactivity, and can induce pleiotropic effects on the bacteria. PMBN at low concentrations is considered to be more specific, as it interacts with LPS to increase permeability, but the effects are dose dependent, and PMBN does have intrinsic growth-inhibitory activity at higher concentrations (67). A more elegant and defined system would be desired for assessment of the role of permeability in compound susceptibility in P. aeruginosa.
Herein we describe the creation of a suite of Pseudomonas aeruginosa mutants in which the final protein involved in transporting LPS for OM biogenesis was compromised in either a wild-type (WT) background or an efflux-deficient mutant. These strains were utilized to assess the activity of multiple classes of antibiotics and to determine the activity-limiting component in P. aeruginosa.
General reagents.All DNA was purified using either the QIAprep spin miniprep kit, DNeasy blood and tissue kit, QIAquick gel extraction kit, or QIAquick PCR purification kit (Qiagen, Valencia, CA). Phusion high-fidelity polymerase and restriction enzymes PstI and BamHI were from New England BioLabs (Ipswitch, MA). In-Fusion HD enzyme mix was from Clontech (Mountain View, CA). Electroporation was performed on a Gene Pulser II electroporator with Gene Pulser cuvettes (Bio-Rad, Hercules, CA). DNA sequencing was performed by Genewiz, Inc. (South Plainfield, NJ). LB, LB agar (LBA), super optimal broth (SOB) medium, cation-adjusted Mueller-Hinton broth (CAMHB), cation-adjusted Mueller-Hinton agar (CAMHA), tryptone, yeast extract, and the BBL Prompt system were from Becton-Dickinson/Difco (Franklin Lakes, NJ). E. coli mating strain RHO3 (68), pEX18Ap plasmid (69), and associated protocols were provided by H. P. Schweizer (Colorado State University). All primers, diaminopimelate (DAP), sodium nitrate (NaNO3), sucrose, EDTA, PMBN, ampicillin, clindamycin, linezolid, colistin, rifampin, tetracycline, ciprofloxacin, novobiocin, fusidic acid, and vancomycin were from Sigma-Aldrich (St. Louis, MO). Gentamicin was from Fisher Scientific (Waltham, MA). Carbenicillin was from Research Products International (Mount Prospect, IL). Azithromycin was from USP (Rockville, MD). Imipenem was from Merck (Kenilworth, NJ).
Pseudomonas strain construction.Mutant alleles lptE6 (30), lpxC101 (25), lptD4213 (28, 70), lptD208 (28, 70), and lptDΔ529–538 (71) were constructed in the suicide vector pEX18Ap (69). Mutations were incorporated into internal primers, which were used to PCR amplify the corresponding gene, in full, in two fragments composed of regions flanking the desired mutation. Upstream and downstream regions were amplified using primer pairs P1/P2 and P3/P4 for lptE6, P5/P6 and P7/P8 for lpxC101, P9/P10 and P11/P12 for lptD4213, P9/P13 and P14/P12 for lptD208, and P9/P15 and P16/P12 for lptDΔ529–538 (Table 1). In a second-round PCR, splicing by overlap extension was used to combine up- and downstream products with external primers P1/P4, P5/P8, and P9/P12 for lptE6, lpxC101, and the lptD alleles, respectively. These complete alleles were then cloned into a BamHI/PstI-digested pEX18Ap vector using In-Fusion HD enzyme. After sequencing to confirm the correct alleles had been made, plasmids were mobilized from E. coli RHO3 (68) into a wild-type P. aeruginosa PAO1 strain termed MB5919 (56) and an efflux-deficient P. aeruginosa PAO1 Δ(mexAB-oprM)::FRT Δ(mexCD-oprJ)::FRT Δ(mexXY)::FRT Δ(mexJKL)::FRT Δ(mexHI-opmD)::FRT Δ(opmH)::FRT mutant termed MB5890 (57) (see Table S1 in the supplemental material). Briefly, overnight cultures of the RHO3 donor grown in SOB medium containing 400 μg/ml DAP and 100 μg/ml ampicillin were diluted 1/100 into 3 ml fresh culture medium and grown to mid-log phase. Overnight cultures of the P. aeruginosa recipient grown in SOB medium were diluted 1:1 in 500 μl 20 mM NaNO3 and incubated at 42°C for 3 h. Both cultures were subsequently washed with 1 ml SOB medium and resuspended in 200 μl SOB medium. Ten microliters of recipient P. aeruginosa cells and 60 μl donor RHO3 E. coli cells were mixed, plated as a dime-sized plane of growth on CAMHA plus 400 μg/ml DAP, and incubated overnight at 37°C. The next day, a loop was dragged twice through the heaviest part of the plane of growth, resuspended in 200 μl CAMHB, and diluted 1/100, and then 70 μl was plated on CAMHA containing either 150 μg/ml carbenicillin (for MB5919) or 40 μg/ml carbenicillin (for MB5890). Exconjugates were confirmed for carbenicillin resistance and sucrose sensitivity. No exconjugates were ever obtained for the lptE6 allele.
To resolve cointegrants to double-crossover mutants, single colonies were restreaked onto LB with no salt (LBNS: 10 g/liter tryptone, 5 g/liter yeast extract) with 10% (wt/vol) sucrose to select for loss of the sacB-containing vector backbone. The resulting colonies were patched onto LBNS, LBNS containing carbenicillin (40 μg/ml for MB5890 and 150 μg/ml for MB5919), and LBNS containing 6 μg/ml rifampin in order to screen for growth, loss of the antibiotic resistance cassette-containing vector backbone, and increased permeability, respectively. Resolved mutants demonstrating the desired Carbs Rifs phenotype were obtained for the lptD4213 and lptD208 mutants, and sequencing across lptD confirmed mutant allele exchange. For the alleles lpxC101 and lptDΔ529–538, hundreds of colonies were screened, and resolved mutants were found to be Carbs but never Rifs. Over 40 resolved clones for each of these constructs were sent for sequencing across lpxC or lptD, respectively, but only wild-type sequence was ever observed.
Unmarked pagP (PA1343) knockouts were constructed in MB5919 and MB5890 using the pEX18Ap suicide plasmid. Flanking regions encompassing 600 bp upstream (through the first 50 nucleotides [nt] of pagP) and 600 bp downstream (including the last 50 nt of pagP) were amplified using primer pairs P17/P18 and P19/P20, respectively, and then subsequently combined through splicing by overlap extension using the external primers P17/P20. The final PCR product was cloned into pEX18Ap, transformed into E. coli RHO3, mobilized into P. aeruginosa, and resolved to the double-crossover knockout as described above. MB5919ΔpagP and MB5890ΔpagP were confirmed by sequencing across the pagP gene.
MIC testing.MICs were determined by the broth microdilution method in CAMHB as recommended by the Clinical and Laboratory Standards Institute (72) in conjunction with the BBL Prompt system. The MIC was scored as the lowest concentration of compounds required to inhibit visible growth after 18 to 22 h of incubation while stationary at 37°C.
Lipid A palmitoylation assay.Overnight cultures were subcultured 1:100 into 5 ml of fresh CAMHB containing 5 μCi/ml 32PO4. Cultures were grown with aeration at 37°C until they reached an optical density at 600 nm (OD600) of ∼1; where indicated, cultures were then treated with 25 mM EDTA for 10 min. Lipid A was isolated as previously described (73). Briefly, cells were pelleted in a clinical centrifuge and washed with phosphate-buffered saline (PBS). Cells were resuspended in a single-phase Bligh-Dyer mixture of chloroform-methanol-water (1:2:0.8 [vol/vol]). Cells were lysed in the mixture for 10 min. The insoluble fraction was collected, resuspended in 12.5 mM sodium acetate (pH 4.5) and 1% SDS, and then incubated at 100°C for 30 min. Lipid A was then isolated from a two-phase Bligh-Dyer extraction and dried under a stream of nitrogen. Samples were resuspended in methanol-chloroform (1:4) mixture, and equivalent amounts were spotted onto a silica thin-layer chromatography (TLC) plate (Macherey-Nagel, Bethlehem, PA). TLC was developed using chloroform, pyridine, 88% formic acid, and water (50:50:16:5 [vol/vol]). Plates were dried and exposed to a phosphor storage screen overnight. Samples were visualized by imaging the screen using a Typhoon scanner (GE, Pittsburgh, PA).
Construction of P. aeruginosa mutants.In order to bridge the gap between in vitro potency and cellular efficacy in P. aeruginosa, matched isogenic mutants of P. aeruginosa were constructed containing mutations putatively leading to permeability defects in a wild-type and multiple-efflux-pump-knockout background. Previous studies have described mutations leading to hyperpermeable (antibiotic hypersusceptibility) phenotypes in E. coli, including mutations in lptD, lptE, and lpxC. Sequence alignment of these genes' proteins from E. coli and P. aeruginosa indicated a fair amount of homology, so five mutant alleles—lptE6 (30), lpxC101 (25), lptD4213 (28, 70), lptD208 (28, 70), and lptDΔ529–538 (71)—were engineered into P. aeruginosa in order to assess whether they would confer similar permeability defects (Fig. 1). Only two of these alleles, lptD4213 and lptD208, were found to be viable. These results held true whether mutants were constructed in the wild-type MB5919 or efflux-deficient MB5890 strain background.
Alignment of LptE, LpxC, and LptD protein sequences from E. coli (Ec) and P. aeruginosa (Pa). Permeability-conferring alleles are listed with the resulting amino acid changes indicated in parentheses, and mutations are illustrated above the sequence with asterisks. Identical residues are highlighted in gray.
Assessing antibiotic susceptibility of P. aeruginosa lptD mutants.To assess the effect of the lptD4213 and lptD208 mutations on P. aeruginosa, strains were tested for susceptibility to a panel of antibiotics. A diverse array of scaffolds comprising a wide array of physicochemical properties were chosen, with compounds spanning a molecular size of 337 to 1,449 g/mol, a calculated value of overall lipophilicity (clogP) of −2.9 to 6.4, a relative polar surface area of 19.5 to 48.3%, 3 to 36 free rotatable bonds, and 8 to 54 hydrogen bond donors and acceptors (74). Antibiotics included classes with activity on Gram-negative bacteria (β-lactam, polymyxin, tetracycline, fluoroquinolone, and aminoglycoside) and those which typically are inactive on Gram-negative bacteria (lincosamide, oxazolidinone, glycopeptide, rifamycin, aminocoumarin, fusidic acid, and macrolide). As can be seen in Table 2, both lptD4213 and lptD208 had a dramatic effect on the susceptibility to several antibiotics. MICs decreased by at least 32-fold for rifampin, ciprofloxacin, tetracycline, novobiocin, and azithromycin in the wild-type MB5919 background, and similar decreases could be observed for fusidic acid and carbenicillin when tested in the MB5890 efflux-null background. Although fold changes in MIC were difficult to assess for vancomycin because activity was not observed at the highest concentration tested (256 μg/ml) in either MB5919 or MB5890, both lptD4213 and lptD208 led to increased susceptibility of the strains and measurable MICs in the range tested. Notably, neither lptD4213 nor lptD208 had significant effects on the susceptibility of P. aeruginosa to gentamicin, clindamycin, or linezolid, with efflux contributing the majority of intrinsic resistance for the latter two. This might be expected given that these three antibiotics are rather small and polar and would not necessarily be subject to exclusion by the LPS barrier of the OM. Taken together, these results indicate that both lptD4213 and lptD208 successfully impart a permeability defect to P. aeruginosa and facilitate penetration of large hydrophobic antibiotics which normally are incapable of traversing the OM of this organism.
In P. aeruginosa, entry of imipenem is facilitated by the OM porin OprD. Altered expression levels (75), changes in amino acid sequence (76), and complete knockout (77) of this protein are frequently associated with carbapenem resistance. It was of interest to know whether the permeability defects imparted by the lptD4213 and lptD208 alleles could compensate for loss of OprD-mediated entry of imipenem. To this end, ΔoprD mutants of MB5890 and MB5919, termed MB6476 and MB6477, respectively, were constructed and subsequently engineered to introduce the two lptD mutations. When imipenem and a control β-lactam that is not specifically transported by OprD, the carbapenem carbenicillin, were tested for activity in these strains, OprD loss had the greatest effect on imipenem, resulting in a 4-fold increase in the MIC whether in an efflux-competent or -deficient background (Table 3). Introduction of either lptD4213 or lptD208 greatly increased susceptibility to imipenem, with resulting MICs of 0.5 to 1 μg/ml, irrespective of efflux capacity, which is equivalent to the MIC range for imipenem in the parental oprD+ MB5890 and MB5919 strains harboring the same permeability defects. Therefore, lptD4213 and lptD208 allow for greater penetration of imipenem than even that afforded by a known substrate-accepting porin.
Furthermore, we sought to compare the permeability defect caused by the lptD4213 and lptD208 alleles to the potentiation achieved with the chemical permeabilizer PMBN. As previously reported, PMBN had little intrinsic activity on the wild-type strain MB5919 (67) but had a MIC of 25.6 μg/ml for the efflux-deficient MB5890 strain. Maximal antibiotic potentiation was essentially achieved with 4 μg/ml PMBN, and this was sufficiently below the MIC for MB5890 to make comparisons. For the most part, MICs in the presence of PMBN (Tables 4 and 5) were within 2-fold of those achieved when strains harbored either the lptD4213 or lptD208 allele (Table 2). The most significant differences were that PMBN potentiated rifampin 16-fold more than either mutant lptD allele in both MB5919 and MB5890, fusidic acid 16-fold more in the MB5919 background, and ciprofloxacin up to 8-fold less in the MB5919 background. This may indicate that PMBN is somewhat better at potentiating certain hydrophobic compounds, but the mutant lptD alleles may be better at potentiating certain hydrophilic compounds.
Assessment of OM integrity in P. aeruginosa lptD mutants.We sought to determine if these lptD mutations caused LPS transport defects in the P. aeruginosa OM. In Gram-negative cells, the OM inner leaflet consists exclusively of phospholipids (PLs), while the outer leaflet is comprised primarily of LPS (78, 79). Mutations that cause defects in LPS transport allow PLs to mislocalize to the outer leaflet and accumulate (41, 80). These mislocalized PLs become available as the substrates of the OM enzyme PagP, which removes the PL sn-1 palmitoyl group and transfers it to the lipid A component of LPS (81, 82). The PagP palmitoylation reaction increases the acylation of LPS, and the presence of such LPS species is used diagnostically to identify compromised OM integrity (41, 80). For example, treatment of cells with EDTA causes loss of LPS from the cell surface, which allows PLs to mislocalize to the OM outer leaflet, triggering PagP palmitoylation of LPS (73).
P. aeruginosa has long been known to palmitoylate its LPS, but only recently has a pagP homolog (PA1343) been identified in PAO1 and shown to function as a palmitoyltransferase (83). We constructed a ΔpagP-null mutant and isolated the 32P-radiolabeled lipid A component of LPS from this strain as well as from the lptD4213 and lptD208 mutants and the wild-type parent. Lipid A species were separated using thin-layer chromatography as described in Materials and Methods. Two major lipid A species were produced by wild-type and ΔpagP strains (Fig. 2). We then isolated lipid A from these strains following treatment with 25 mM EDTA. Two novel lipid A species were produced by the wild type in response to EDTA treatment but were not produced by EDTA-treated ΔpagP cells. Clearly, the appearance of these novel lipid A species is PagP dependent, and these therefore represent palmitoylated lipid A species. The same PagP-modified lipid A species are highly abundant in lptD4213- and lptD208-harboring P. aeruginosa strains without the need for EDTA treatment (Fig. 2), confirming that the organization of the OM lipid bilayer is severely disrupted in these mutant strains.
Mutations in P. aeruginosa lptD cause cells to produce LPS palmitoylated by PagP. LPS was extracted from stationary-phase cultures of MB5890 as well as ΔpagP, lptD208, or lptD4213 derivative strains that were grown in CAMHB and radiolabeled with 32P. Where indicated, cultures were treated with 25 mM EDTA for 15 min before sample collection. Samples were developed by TLC and visualized using a phosphor screen. The two major lipid A species produced in wild-type cells are denoted as "wt." The two major PagP-dependent lipid A species are marked with asterisks.
Despite the need for novel Gram-negative antibiotics, new classes with alternative mechanisms of action have not been forthcoming. Failure can often be attributed to an inability to achieve cellular activity with target-based in vitro inhibitors, perhaps caused by initial hits occupying the wrong physicochemical property space (13, 14, 74). When there is success in attaining cellular bioactivity, close attention must be paid to overcoming intrinsic resistance mechanisms such as efflux and permeability. Such efforts have shown promise in recent years with the development of hydroxamate LpxC inhibitors (84–86), novel bacterial type II topoisomerase inhibitors (NBTIs) (87, 88), tricyclic GyrB/ParE (TriBE) inhibitors (89), and pyrrolocytosine ribosome inhibitors (90). Although these classes of compounds have not yet reached clinical utility, they demonstrate progress and inspire confidence that novel Gram-negative antibiotics can be discovered.
One of the tools missing for assessment of determinants of compound activity in the major pathogen P. aeruginosa is a defined genetic means of measuring the contribution of permeability to intrinsic resistance. An antibiotic-hypersusceptible mutant, termed Z61, has been used to assess the effect of increased permeability on compound activity in P. aeruginosa (91–93); however this mutant was derived through multiple rounds of mutagenesis and contains hundreds of single nucleotide polymorphisms (SNPs) (94). Since numerous suppressors could be isolated with a spectrum of partial to full phenotypic reversion (91) and independent mutations could be identified leading to supersusceptibility to subsets of antimicrobial agents (95)—including oprM, ampC, lptE, and amgRS (94)—the Z61 strain is undoubtedly genetically complex, and hypersusceptibility cannot be solely attributed to defects in permeability. Additionally, the E. coli porin OmpF has been introduced into P. aeruginosa, resulting in strains significantly more susceptible to penem antibiotics (96). Given that porin expression was driven off a plasmid, results could vary due to expression level, and increased permeability is likely limited to smaller hydrophilic molecules.
Herein, we have created defined single mutations which lead to a general permeabilization of P. aeruginosa. This approach allows construction of isogenic strain sets that can separate the effects of compound influx from efflux and guide compound design to address the activity-limiting factor. The two mutations described herein, lptD4213and lptD208, are overlapping deletions in LptD, a β-barrel protein responsible for the final step of LPS transport and assembly in the OM (40, 41, 70, 71). Based on recent crystal structures (97, 98), both mutations lead to the deletion of the alpha-helical loop L4 situated on the extracellular surface of the protein, as well as a portion of either β7 or β8, respectively. Deletion of this loop may impair LptD function by reducing a few hydrogen bond interactions with the LptE plug and increasing extracellular access to the lumen of the β-barrel. Interestingly, one of the attempted mutations that was found to be nonviable, lptDΔ529–538, is also in LptD. However, this mutation leads to deletion of a large loop, L8, between β14 and β15, which extends much further into the lumen of the LptD β-barrel and makes extensive interactions with LptE. As was similarly suggested by the lptE6 mutation, which was found to be dominant negative, the interactions between LptD and LptE may be particularly critical in P. aeruginosa, and significant disruption may be lethal.
In both the wild-type MB5919 and efflux-deficient MB5890 backgrounds, the lptD4213 and lptD208 alleles were demonstrated to impart significant permeability defects, as assessed by increased susceptibility to a range of antibiotics. The power of these defined genetic lesions and the use of isogenic strain sets are exemplified by the ability to define the activity-limiting component for the antibiotics tested. We determined that efflux was the major contributor to intrinsic resistance for clindamycin and linezolid, with the OM barrier providing minimal protection. Conversely, imipenem, vancomycin, colistin, and rifampin were found to be poor substrates for efflux, and their activity was mostly limited by poor entry into P. aeruginosa. For ciprofloxacin and tetracycline, efflux was the main factor limiting activity as lptD4213 or lptD208 mutations did not sensitize MB5890. However, in the wild-type MB5919 background, introduction of the lptD4213 and lptD208 alleles did reduce the MICs between 16- and 32-fold, indicating that increased penetration of ciprofloxacin and tetracycline into P. aeruginosa could overwhelm the efflux pumps and increase their bioactivity. For novobiocin, carbenicillin, fusidic acid, and azithromycin, both efflux and the OM barrier function contribute to intrinsic resistance, as mutations in either lead to additive improvements in potency. Finally, for gentamicin, neither efflux nor permeability appears to be a limiting factor for activity, as mutations in either did not significantly increase the susceptibility of P. aeruginosa.
LPS palmitoylation by PagP in strains harboring lptD4213 and lptD208 demonstrated that these alleles cause defects that disrupt the lipid asymmetry of the OM bilayer. Similar defects resulting in PagP-modified LPS can be observed upon depletion of the LptD and LptE proteins in E. coli (41, 80). The LPS perturbation observed in these mutants genetically phenocopies the results observed by chemical perturbation using reagents such as EDTA. As such, these strains will be extremely useful in antibacterial discovery to track activity of weakly bioactive compounds and to optimize hits from both phenotypic and biochemical screens.
In addition to being useful tools for delineating the factors affecting susceptibility to particular compounds, these permeability mutants will be useful in understanding basic biology related to outer membrane protein (OMP) assembly and LPS transport. Indeed, in E. coli, chemical conditionality was used in conjunction with the lptD4213 allele to screen for suppressors of vancomycin (and related analogs) susceptibility, leading to identification of several components of the β-barrel assembly machinery, including bamA (99) and bamB (100, 101). Furthermore, it was the initial E. coli lptD4213 strain that was used to determine that LptD was involved in OM biogenesis (70) and ultimately responsible for LPS transport (39–41). To date, there have been limited studies exploring P. aeruginosa β-barrel assembly and the interplay with LptD function and LPS transport. As this study implies, the inability to engineer certain lptE and lptD alleles in P. aeruginosa that are viable in E. coli suggests that differences in the function of the LPS transport and β-barrel assembly machinery exist between organisms. This is further supported by the discovery of an extremely potent P. aeruginosa-specific LptD inhibitor that shows no activity on other Gram-negative organisms (102, 103). Clearly these systems are of importance and represent valuable targets for the development of novel antibiotics.
Returned for modification 26 August 2015.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01747-15.
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. 2010. Screening for antibacterial inhibitors of the UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) using a high-throughput mass spectrometry assay. J Biomol Screen 15:52–61. doi:10.1177/1087057109355319.
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. 2009. New antibiotic molecules: bypassing the membrane barrier of gram negative bacteria increases the activity of peptide deformylase inhibitors. PLoS One 4:e6443. doi:10.1371/journal.pone.0006443.
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. 2010. Peptidomimetic antibiotics target outer-membrane biogenesis in Pseudomonas aeruginosa. Science 327:1010–1013. doi:10.1126/science.1182749.
. 2012. Inhibition of lipopolysaccharide transport to the outer membrane in Pseudomonas aeruginosa by peptidomimetic antibiotics. Chembiochem 13:1767–1775. doi:10.1002/cbic.201200276.


"The still image in this video was created to fit with the theme of the song, Lent the Hand. The coming together of the two hands, from unknown beings, portrays the idea of lending and accepting a helping hand, not only to give but also receive."
Lent the Hand featuring Ned Singh on vocals blends elements of Electronic to Progressive House. It is fire and ice with energetic transients, and smooth melodies. It sends off a triumphant vibe and keeps one engaged in battle.
An Ordinary day, from Houston, brings together a fusion of smooth, soulful, and edgy vibes that capture the soundtrack of life. Drawing a diverse taste and take on music, An Ordinary Day takes the listener on a journey through use of multiple production elements that give a distinct, raw, and organic sound. The influence of various styles serves a melting pot of music, that meshes the real and ideal.
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Youngsters attending Eaton Socon Cricket Club's Friday fun junior sessions are pictured with back row, from the left, sponsor Matt Giggs and coaches Matt Taylor, Graeme Banks and Robert Ashwell.
Eaton Socon Under 15s booked their place in the Hunts County Cup final with an eight-wicket win over Huntingdon & District.
Scott Stevenson and Joe Watling both took two wickets as Huntingdon & District were restricted to 95-8 before easing past their target. Dan Wood hit an unbeaten 51 (a batsman must retire when at 50) to set up a final against either Ramsey or Peterborough Town.
Eaton Socon run Friday-night fun sessions at Peppercorns Lane – 4.30pm to 6pm for Under 9s and 6.30pm to 8pm for Under 10s to Under 15s. The cost is £3.


Business communication, in its writing business correspondence and documentation and oral business speech forms is highly conventional, that is, it strictly follows a number of generally accepted norms and rules, as a voice plan, and then we are talking about speech etiquette, so and a behavioral one business etiquette. Persuasion is an impact through the evidences, logical ordering of facts and conclusions. She also goes on to say that continuous feedback and coaching provides an opportunity for solid communication between a supervisor and employee, and should be kept to a simple format, and as an informal conversation when it's not in the pretext of a formal review or personnel action. For that sample teacher cover letter for job it is important for a manager to understand how to deal with the personal barriers that different employees may have to communicating.
Communication c. Allowing the other person to say what they have in mind without interruption assures the listener of having all the facts before any conclusions or assumptions are reached. Thus, it is necessary to consider that may be tensions and even conflict situations in the process of communication between partners and colleagues.
Davep. Business communication exists in two forms: There are many forms of unspoken communication.
A manager must work on developing his or her listening skills if he or she wishes to be able to connect with his or her staff. What is more, we are how to make a counter argument in an argumentative essay about the interests of individuals and entities. While often modern technology can help eliminate noise barriers, sometimes noise barriers cannot be helped.
While often modern technology can help eliminate noise barriers, sometimes noise barriers cannot be helped. The power of example plays an important role in suggestion that causes conscious copying of behavior and unconscious imitation.
Date submitted: Taking responsibility to small roofing contractor business plan the issue of miscommunication is describe your future husband essay to ensuring the lines of communication are open and free of uncertainties.
If the general public were to become aware of a new product, a new acquisition or some other similar type of big information can impact the morale of the staff.
To echo what the management has stated does not show the employee that a supervisor will stand up for their concerns. Different communication barriers have different solutions.
A wide range of business firms found that their employees were ill equipped for overseas work in the globalizing market. We're also more essay on solar energy to find these types of employees less likely to get caught up in corporate gossip or rumor mills and promoting more communication disconnect.
Process Barriers: If someone becomes upset, then the intended message is blurred with the raw emotion of human nature. The spread of information, through technical communication, is given more accurately and is made easier for the reader to understand.
Furthermore, an unskillful use of any word can lead to considerable information losses, and, hence, to failures in the performance of official duties. Poor employee to supervisor communication can also lead to higher tardiness, absenteeism and other detrimental results from lack of communication.
The various factors, including nature, content and communication situations normal, extremea public or official position authority and personal qualities of the subjects of communication, essay writing in business communication an impact on the choice of the methods of influence people. Therefore, business communication is a necessary part of human life, the most important type of relations with other people.
Thinking about the different ways we communicate with essay writing in business communication. Multiple choice: Semantic Barriers: Family businesses often have to struggle with conflicts among members of the organization, low managerial abilities, interfered daily business activities, and customer relationships that suffer from the lack of structure… Business Communication Words 5 Pages Assignment f SUBJECT Business communication Tutorial group: The obvious comes to mind: Emotional barriers cannot be ignored.
Not only can the use of wrong words be confusing but the right words should be used when communicating information to the right people. It presents the concept of business communication, its main forms and methods, and effective practices that are necessary for effective business communication.
When the sender of information uses the wrong words to essay writing in business communication their message, the receiver may not be able to understand. Specialized business knowledge is important, but not enough to guarantee success. In addition to perception barriers and listening barriers, psychological distance barriers can hamper the transferring of business plan graham balls within an organization.
This system is regulated through feedback, that is, equivalence of this situation adopted by two communicants.
Additionally, the sender of information should make sure that he or essay writing in business communication is not making the information more complicated by presenting it in a confusing manner. External Communications 16 Table 5: Just complete our simple order form and you could have your customised Business work in your email box, in as little as 3 hours.
Discussion of this question has been arranged as follows: We speak, write, walk, make facial gestures, and many other subtle and not-so-subtle body language queues that communicate information to those around us in the workplace.
By utilizing this technique, you'll demonstrate that you were paying attention.
Informal types of oral communication are discussions that take place in a business meeting,… Communication Is The Most Effective Form Of Business Communication Words 4 Pages environment communication is major factor. While perception barriers, listening barriers and psychological distance barriers are harmful one of the most detrimental kinds of barriers are emotional barriers.
This type of unspoken communication shows the employee he is valued and trusted. All to often these diverse entities within a company can deliver opposing messages, confusing employees and supervisors. In this case study, Molly and Daniel found that there was a problem in communication between… Communication Problems in Family Business Words 6 Pages 'Communication Problems in the Family Business ' Abstract For family businesses, effective communication is an even more integral factor for successful business operations equity research cover letter wso to the peculiarity business plan graham balls personal family emotions frequently interfere with business decisions.
There are UK writers just like me on hand, waiting to help you. Having a sense of professionalism will bring a lot to the business, especially in a long term relationship with employees and clients. Order Now The Importance of Business Communication When you think about these two quotes you will find a lot of meanings, something happens in your life around essay writing in business communication, about the relations and communication and how to deal with others not just on business also in your general life, so it is too important to learn how to communicate.
Let us start to examine the many facets essay writing in business communication effective communication in the workplace. Law enforcement officials use interrogation techniques designed to elicit involuntary body language responses to determine trustworthiness of the information being given.
Volume, inflection, facial expression, and concurrent body language all set the combined tone of the conversation taking place. One of the most common adages in human nature is "no news is good news" but in corporate communication, that statement could not be further from the truth.
Physical barriers is another category of communication barriers. Technical communication is the dispersal of information that would aid in the way people interact with technology. Instead, a person who wishes to work past emotional barriers must understand the barrier and work towards removing the barrier.
If a supervisor cannot find the answers going through the channels and gives conflicting information to the employee, the gap in information then will propagate throughout the workforce.
Workplace stress is commonplace in the twenty first century.
It is key for a business to be o que significa a palavra business plan to communicate properly, with such examples, setting daily tasks for the employees of the company. The employees are responsible for being better prepared and for proactively working with supervisors in times where questions arise.
The use of good communication within ones business is vital in order to maintain a fully functional and co-operative company. Transferring a message from a sender to a receiver is defined as communication. Why Good Communication is good business. For that reason it is important for a manager to understand how to deal with essay writing in business communication personal barriers that different employees may have to communicating.
Physical Barriers: Jargon is language that is used in a specific field. One of the main regulators of these relations are the ethical norms that are expressed in our concepts of good and evil, justice and injustice, right equity research cover letter wso wrong actions of people.
Effective business communication requires effective practices Essay | Expert Writers A customer who receives poor communication from an employee will most likely have an unfavorable response to a company. As long as the supervisor keeps some of these thoughts in mind when having a talk with an employee, the supervisor can control the conversation in most every way.
Once the person has calmed down, go to them and ask them how you could avoid having the situation in the future. Effective communication can also improve relationships at work, home, and in social situations by deepening your connections to others and improving teamwork, decision making, caring, and problem solving. Oral Communication is the process of verbally transmitting essay writing in business communication give and exchange information to another person or people.
Paying attention to the speaker with eye contact, gestures of nodding of the head and similar physical responses to provide feedback that you are listening is a first step in avoiding miscommunication. Tesco staff meeting to organise a fundraiser for a nearby charity raising money for Cancer research.
Oral communication can be formal or informal.
Take the time to see how an how to make a counter argument in an argumentative essay works and reacts when given criticism, and see how the employee reacts and works when given praise. Globalization essay css pdf are two forms of communication: One of the techniques to better communication is small roofing contractor business plan start with active listening.
Canned, impersonal recognition events are not effective. Every level of employee should understand how the hierarchy works and to whom to direct their questions.
Be visible, make yourself easily found and approachable. Employees are also expected to have prepared for meetings, essay writing in business communication reviews, and informal conversations with others.
Business is all about communicating efficiently. What are barriers and break-downs in communication? Those who have poor listening skills will often misunderstand or misinterpret the information being describe sample teacher cover letter for job future husband essay. Related Essays. For Group Assignment only. How to cite this page Choose cite format: Often overlooked is the reverse communication relationship between the employee and supervisor.
An employee who feels that he may be the last to have information may feel less ice essay group than his peers may. However, international business communications do not effective every time.
Many times the HR department walks a tightrope between being an employee advocate, and being a consultant and good steward for the company. A team that has good listening skills will be one that can communicate with each other effectively.


text:
namespace System.Windows.Forms
{
    public struct Message
    {
        public IntPtr HWnd { get; set; }
        public int Msg { get; set; }
        public IntPtr WParam { get; set; }
        public IntPtr LParam { get; set; }
        public IntPtr Result { get; set; }
        public object GetLParam(Type cls) => null;
        public static Message Create(IntPtr hWnd, int msg, IntPtr wparam, IntPtr lparam) => default(Message);
        public override bool Equals(object o) => false;
        public static bool operator !=(Message a, Message b) => false;
        public static bool operator ==(Message a, Message b) => false;
        public override int GetHashCode() => 0;
        public override string ToString() => "";
    }
}

redpajama_set_name: RedPajamaGithub


Absinthe is formidable alcoholic liquor manufactured from alcohol and different types of herbs. The herbs are either distilled or herbal extracts are utilized to prepare absinthe. The chief herbs used are wormwood (Artemisia absinthium), anise, melissa, hyssop, and fennel. Often calamus and nutmeg were also employed. Absinthe was invented at the end of the eighteenth century by a French doctor Dr Pierre Ordinaire in Switzerland. It was primarily utilized as a digestive tonic but shortly its mystical effects achieved it the Numero Uno drink of nineteenth century Europe. It had become lovingly called as the "Green Fairy "or "Green Muse".
The term absinthe conjures up images of the grand cafés, the European masters, Moulin Rouge, Parisian theatres and all the other symbols of the Belle poque. It was a common sight to discover great number of people drinking absinthe in the French bars, cafés and bistros. Evening 5 p.m. was regarded as the Green Hour or"Heure Verte". Absinthe was at its peak reputation throughout the period 1880-1910; it was during this time period that it rivaled wine. Description of Belle poque French society will be incomplete devoid of discussing absinthe. Not any other country in Europe utilized absinthe in quantities comparable to France. Absinthe or the green fairy as it was called was considered an inspirational drink and several artists and writers who revolutionized art and literature in Europe ascribed their imaginative genius to the absinthe green fairy.
So, what was it that made absinthe so special? Well, absinthe is unlike any other drink. Its content has a very high percentage of alcohol and extracts of herbs the main herb being wormwood. Wormwood includes thujone which happens to be responsible for the unique effects of absinthe. Wormwood herb is closely related to the daisy family and grows in several areas of Europe. Thujone is a brain catalyst and its chemical structure is similar to THC or Tetrahydrocannabinol which happens to be present in cannabis.
Absinthe that is carefully distilled by the very best distilleries just isn't very bitter. Nevertheless, absinthe created by many of the less popular distilleries is extremely bitter. It has anise and this provides it with the licorice taste. Absinthe also has a small taste of citrus.
Absinthe is an extremely liberating drink and an evening with the Green Fairy completely rejuvenates the body and mind. Absinthe drinking is a ritual and there are two kinds of rituals you'll be able to decide on to prepare your absinthe drink. The standard French ritual and a modern Czech ritual made well-known by the film "Moulin Rouge". Some of the very famous absinthe drinkers mentioned Vincent Van Gogh, August Strindberg, Arthur Richmond, Oscar Wilde, and Pablo Picasso.
Regardless of what ritual you determine to ready your absinthe drink, apart from absinthe additionally, you will need absinthe glass, and absinthe spoon. Absinthe spoons and glasses can be found in various styles. Quite a lot of history and culture is associated to them. A few of the pre ban antique absinthe, spoons and glasses demand a hefty premium in the antique market.
For most of the 20th century absinthe was banned in most of Europe and the United States. This was on account of great misconceptions concerning absinthe that were in currency in those days. However, since the beginning of the century most European countries have lifted the unfair ban and absinthe has returned with a bang. The internet has made it very easy for lovers of absinthe to find the best quality absinthe online.
You can order your chosen absinthe online from many of the most recognized names in the industry. Buying absinthe and also other absinthe products online is fairly easy and safe. Visit absinthekit.com and put your orders for real absinthe essence and ready your own absinthe at home.
So, go and acquire your absinthe and spend a wonderful evening with the absinthe green fairy or the green goddess.


text:
HIV Testing Should Require Consent and Promise Privacy
This article appeared in the San Francisco Chronicle on June 6, 2007.
The lure of federal dollars may be prompting California lawmakers to consider an ill-advised law to make HIV testing routine. But there's nothing routine about testing for HIV, and legislative efforts to make it more common show little concern for the frankness of the doctor-patient relationship, or the insidious threats to patient privacy.
Assembly member Patty Berg, D-Eureka, introduced a bill in February to encourage routine testing for HIV by eliminating state requirements that patients provide written consent to the procedure. The bill was voted out of the Assembly Appropriations Committee on May 31 and is expected to come to a vote before the full Assembly later this week and possibly as early as today.
No lawmaker admits as much, but the effort may, at least in part, be driven by the new federal Ryan White Grant Program requirements that states provide the names of HIV patients — not just anonymous numbers — to the federal government in order to receive funding for state HIV programs. To maximize numbers, the bill absolves physicians of any need to obtain written consent or do pre-test counseling, and creates a presumption that the test will be done unless the patient specifically objects.
The U.S. Department of Health and Human Services announced late last month that three California counties (San Francisco, Marin and San Mateo) will lose $8.6 million in federal funds under the Ryan White program this year, as funds are redistributed from urban to rural areas.
San Francisco was hit especially hard, so Sacramento lawmakers no doubt hope to identify as many patients with HIV as possible, because greater numbers mean more funds. If the law passes, the net result will be that patients are tested with no legal requirement that they've received an adequate explanation of the associated risks. In essence, the law would sweep the "informed" part of "informed consent" under the rug.
No medical care should be so routine that physicians are dissuaded from discussing with patients their individual needs and concerns. A patient should only be tested for HIV if and when a physician determines that it's appropriate in view of the patient's particular circumstances. Unnecessary tests are against the patient's best interests, are likely to create mistrust and could discourage people who might need to be tested from seeking crucial medical care.
Furthermore, when doctors decide testing is appropriate, their patients need to know about the various testing options, the chance of false positives and the parties to whom the results will be disclosed. These are the sorts of things doctors discuss with their patients during the informed consent process. The only logical reason to eliminate counseling and written consent is a fear that the knowledge learned might dissuade patients from consenting in the first place.
The California bill states that documentation and disclosure will be made in accordance with existing state law, but how is that confidentiality to be assured if the Ryan White program requires states to identify HIV/AIDS patients by name? The answer is that it won't. In April, Gov. Arnold Schwarzenegger signed into law a bill sponsored by Sen. Nell Soto, D-Pomona, that eliminated the state's confidentiality protections. No more anonymous privacy codes, just each patients name clearly printed on the HIV test results.
Loss of confidentiality is a real risk, and a real concern. Florida implemented the Ryan White requirements early, and in 2005 a Palm Beach health department employee inadvertently sent 800 employees an internal e-mail containing a list of about 6,500 HIV and AIDS patients. Once the cat was out of the bag, it is hard to know how many of those 800 employees shared what they had learned with spouses and friends or even others.
Reports of HIV patients suing their doctors for inadvertent disclosure to insurance companies and employers are far from uncommon. The fact that the disclosures are illegal is beside the point. The sheer volume of data that doctors need to report to state and federal agencies all but assures such blunders will occur.
The easiest way for states to get as many names as possible to meet the new Ryan White requirements is to make HIV test results a routine entry in patients' charts, whether or not patients understand the consequences of being tested or not, whether physicians believe the tests are necessary or not and whether such testing is even in a patient's best interest or not.
HIV testing is not like cholesterol testing. People don't worry about their cholesterol levels becoming public or about being stigmatized for their cholesterol status. Federal and state governments introduced confidentiality rules and anonymous testing policies to reassure patients that their privacy would be respected, in hopes of encouraging them to choose freely to risk the loss of privacy and get tested. Eliminating privacy protections and testing routinely could cause those who most need to be tested to avoid medical treatment altogether.
It's bad enough for a state government to decide that it wants to protect people from themselves by making medical choices for them. It's all the worse when that supposedly well-intentioned paternalism is really a ploy to dip into federal coffers.


text:
Leeds cyclist to travel across Vietnam and Cambodia for St George's Crypt charity
Joseph Keith
Published: 06:00 Saturday 17 November 2018
He will take in some of the world's most-stunning landscape views, passing through centuries of history along his quest in aid of charity - all while travelling on a bicycle.
Fundraiser Gary Hudson is now preparing to saddle up to take part in a cross-country cycling challenge through Vietnam and Cambodia.
Gary Hudson is preparing to cycle across Vietnam and Cambodia. Picture: Tony Johnson.
The 50-year-old, from Middleton, will begin his journey tomorrow and aims to finish by November 27 - ten days later.
After self-funding the trip to Asia, the dad-of-two has already raised more than £2,000 for the St George's Crypt homelessness charity in Leeds.
"I have always been passionate about helping the homeless and always wanted to go to Vietnam and Cambodia ever since I was a young child," he told the YEP.
"I have been volunteering down at St George's Crypt, going behind the scenes.
"I've been able to see what they do and all the great projects they do for the homeless and vulnerable people of Leeds, they do so much work and a fantastic job.
"There's so many great things that they do and I hope that this will help raise awareness as well as funds."
The keen cyclist set off on his flight from London today (Saturday) and will on Sunday arrive in Ho Chi Minh City, also known as Saigon, via Singapore.
His gruelling cycling journey begins on Monday, when he leaves the Vietnamese city in the direction of Tra Vinh.
On the fifth day of the challenge, Mr Hudson then plans to take a boat, led by a guide, from the edge of Vietnam to Phnom Penh, neighbouring Cambodia's capital.
During the journey, he will also visit the Cambodian Killing Fields, where more than one million people were killed and buried following the end of the nation's civil war period.
Other prominent locations that he will see include the Angkor Wat temple complex and world heritage site at Siem Reap, in Cambodia, before flying back to England.
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Mr Hudson, who works on Barclays' customer experience team, said he has stepped up his training ahead of the challenge.
"I do a lot of cycling but I have been doing a lot of extensive training," he said.
"I've been taking myself off on longer rides, of 60 or 70km, jsut to get myself used to it, physically and mentally, staying in the saddle for such a long time.
"I can't prepare for the heat though and it's going to be very hot and humid out there."
He said the support he had received from loved ones had been invaluable, and plans to blog about his experiences throughout the challenge while abroad.
"I have never done anything like this before," he said.
"I feel quite privileged to be able to take this opportunity and do something for such a great cause.
"My family, friends and colleagues have all been right behind me with their support, too."
Click here to follow Mr Hudson's blog or donate to his fundraising
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When new TVs or mobile phones sit on store shelves, they seem self-contained. The experience is designed to inspire awe: innovations are placed on shrine-like displays with no signs of their manufacturing; all finished, polished, and gift-wrapped in plastic; waiting to be taken home. But if you look under the cover of any innovation, the magic of self-containment fades. There are subinventions, subproducts, minor-breakthroughs, and parts and components, each with a story of their own. Every wondrous thing is comprised of many other wondrous things.
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